Human eosinophil-derived basic protein

ABSTRACT

The present invention provides a human eosinophil-derived basic protein (EBPH) and polynucleotides which identify and encode EBPH. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding EBPH and a method for producing EBPH. The invention also provides for use of EBPH and agonists, antibodies or antagonists specifically binding EBPH, in the prevention and treatment of diseases associated with expression of EBPH. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding EBPH for the treatment of diseases associated with the expression of EBPH. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding EBPH.

This application is a divisional application of U.S. application Ser.No. 08/740,036 , filed Oct. 23, 1996.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of anovel human eosinophil-derived basic protein and to the use of thesesequences in the diagnosis, prevention, and treatment of disease.

BACKGROUND OF THE INVENTION

Stem cells are progenitor blood cells which differentiate to maturewhite blood cells, red blood cells, and platelets. Stem cells are foundin adult bone marrow, in fetal liver and spleen, and in blood collectedfrom the umbilical cord after the birth of a baby.

Eosinophil growth and differentiation from stem cells is regulated byhematopoietic growth factors including granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5(IL-5). IL-5 is a potent eosinophil differentiation and activationfactor, while GM-CSF and IL-3 also increase the production of othermyeloid cells.

Eosinophils are white blood cells which are sub-classified asgranulocytes due to the presence of large, coarse membrane-boundcytoplasmic granules. These granules contain proteins and othercompounds which carry out a variety of inflammatory and immunefunctions. In response to chemotactic factors, eosinophils migratethrough blood vessel walls and through tissues to the site where theyare needed. There the contents of the granules are released in responseto specific stimuli. Eosinophil granule release is stimulated byimmunoglobulin E (IgE)-mediated hypersensitivity reactions such asparasitic infections and type I allergic reactions. Such type I allergicreactions include asthma and allergic rhinitis.

A variety of eosinophil-derived basic proteins (EDBPs) are released fromeosinophil granules. These cytotoxic proteins disrupt membrane surfacesand lyse cells. EDBPs are thus potent anti-parasitic and anti-bacterialagents, however, EDBPs may also damage host tissues. For instance, thecardiovascular damage associated with chronic hypereosinophilia has beenattributed in part to secreted EDBPs. EDBPs have been shown to damagerespiratory epithelial cells, and have been implicated in the increasein bronchial hyperreactivity frequently observed in asthma patients. Asignificant correlation exists between the intensity of bronchialhyperreactivity and the levels of EDBPs in blood and bronchoalveolarlavage (BAL) fluid from asthmatics.

Eosinophil granule major basic protein (MBP), one of the mostextensively characterized EDBPs, is a potent toxin against helminths,protozoa, bacteria and other cells. MBP also causes epithelialdesquamization and ciliostasis, effects that mimic the pathology ofasthma (Gleich, G. J. et al. (1988) J. Allergy Clin. Immun. 81:776-781).MBP is found in sputa and on damaged bronchial tissues of asthmapatients (Frigas, E. et al. (1981) Mayo Clin. Proc. 56:345; Filley, W.V. et al. (1982) Lancet 2:11).

Along with its cytolytic properties, MBP also possesses noncytolyticproinflammatory properties, many of which are associated with late phasereactions of allergic disease. The release of histamine and leukotrieneC4 from basophils is stimulated by MBP and further enhanced by thecytokines IL-3, IL-5 and GM-CSF (Sarmiento, E. U. et al. (1995) J.Immunol. 155:2211-2221). MBP also stimulates neutrophil activation anddegranulation, including the release of superoxide anion (O2-) andlysozyme (Moy, J. N. et al. (1990) J. Immunol. 145:2626-2632), andplatelet activation (Rohrbach, M. S. et al. (1990) J. Exp. Med.172:1271-1274). MBP also induces further eosinophil degranulation (Kita,H. et al. (1995) J. Immunol. 154:4749-4758).

The cDNA for MBP encodes a 25 kdal preproprotein molecule of 222 aminoacids, which includes a predicted 15 amino acid leader peptide, a 90amino acid acidic pro domain and a 117 amino acid mature polypeptide(Popken-Harris, P. et al. (1995) J. Immunol. 155:1472-1480). The prodomain of MBP contains a heterogeneous population of O- and N-linkedglycosyl modifications and has an isoelectric point (pI) ofapproximately 4.9. The 14 kdal mature MBP contains two disulfide bridgesand five free cysteine residues (Oxvig, C. et al. (1994) FEBS Lett.341:213-217).

The negatively-charged pro domain appears to interact with thepositively-charged mature MBP. This interaction is proposed to inhibitthe activity of mature MBP thus protecting the developing eosinophilfrom damage by MBP during granule processing (Popken-Harris, et al.(1995), supra). Mature MBP, but not proMBP, reacts readily with acidiclipids and disorders lipid bilayers resulting in the lysis of liposomes(Abu-Ghazaleh, R. I. et al. (1992) J. Membr. Biol. 128:153-164). Unlikemature MBP, proMBP does not stimulate basophil histamine release orneutrophil superoxide generation; in fact, proMBP is an inhibitor ofthese MPB-stimulated activities (Popken-Harris, et al. (1995), supra).

ProMBP is expressed in placental X cells and is found in the sera ofpregnant women (Wagner, J. M. et al. (1994) Placenta 15:625-640). Levelsof proMBP peak before labor and rapidly decline after delivery (Maddox,D. E. et al. (1983) J. Exp. Med. 158:1211-1216). ProMBP of placentalorigin is heavily glycosylated and circulates in disulfide-bridgedcomplexes with pregnancy-associated plasma protein A (PAPP-A),angiotensinogen, and complement C3dg (Oxvig C. et al. (1993) J. Biol.Chem. 268:12243-12246; Oxvig C. et al (1995) J. Biol. Chem.270:13645-13651). Low serum levels of PAPP-A in the first trimester havebeen linked to fetal chromosomal abnormalities (Brambati, M. C. (1993)Br. J. Obstet. Gynaecol. 100:324-326). A high molecular weight (HMW)form of angiotensinogen has been found in moderate quantities in plasmafrom pregnant women and in high quantities in hypertensive pregnantwomen (Tewksbury, D. A. et al. (1989) Am. J. Hypertens. 2:411-413).Oxvig and colleagues (1995, supra) suggest that this HMW angiotensinogenis actually the proMBP:angiotensinogen complex.

The discovery of polynucleotides encoding a novel EDBP-like molecule,and the molecule themselves, satisfies a need in the art by providing anew means for the diagnosis, prevention, or treatment of diseases andconditions associated with eosinophil accumulation and granule releaseincluding late-phase allergic/inflammatory reactions, eosinophilias,parasitic infections, and conditions associated with placentalderived-EDBP accumulation in pregnancy.

SUMMARY OF THE INVENTION

The present invention features a novel basic protein derived from IL-5cultured umbilical cord blood cells, hereinafter designated as EBPH andcharacterized as having chemical and structural homology to eosinophilgranule MBPs from human and guinea pig.

Accordingly, the invention features a substantially purified human EBPHhaving the structural characteristics of the MBPs above and as shown inamino acid sequence, SEQ ID NO:1.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode EBPH. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

The invention also features a polynucleotide sequence comprising thecomplement of SEQ ID NO:2 or variants thereof. In addition, theinvention features polynucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention additionally features nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode EBPH. The present inventionalso features antibodies which bind specifically to EBPH, andpharmaceutical compositions comprising substantially purified EBPH. Theinvention also features agonists and antagonists of EBPH.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A 1B, and 1C show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of EBPH. The alignment was producedusing MacDNASIS PRO™ software (Hitachi Software Engineering Co., Ltd.,San Bruno, Calif.).

FIGS. 2A and 2B shows the amino acid sequence alignments among EBPH (SEQID NO:1), eosinophil granule MBP from human (GI 34476; SEQ ID NO:3) andtwo MBP homologs from guinea pig, GMBP-1 (GI 220291; SEQ ID NO:4), andGMBP-2 (GI 544241, SEQ ID NO:5). The alignment was produced using themultisequence alignment program of DNASTAR™ software (DNASTAR Inc,Madison Wis.).

FIG. 3 shows the hydrophobicity plot (MacDNASIS PRO software) for EBPH,SEQ ID NO:1; the positive X axis reflects amino acid position, and thenegative Y axis, hydrophobicity.

DESCRIPTION OF THE INVENTION

Before the present protein, nucleotide sequence, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,"amino acid sequence" as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence and fragments or portions thereof, of anaturally occurring or synthetic molecule.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "protein" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Peptide nucleic acid", as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

EBPH, as used herein, refers to the amino acid sequences ofsubstantially purified EBPH obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GCGFragment Assembly™ system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A "variant" of EBPH, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic EBPH, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to EBPH, causes a change in EBPH which modulates the activity ofEBPH. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to EBPH.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to EBPH, blocks the activity of EBPH.Antagonists and inhibitors may include proteins, nucleic acids,carbohydrates, or any other molecules which bind to EBPH.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of EBPH. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of EBPH.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of EBPH orportions thereof and, as such, is able to effect some or all of theactions of EBPH.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding EBPH or the encoded EBPH.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. et al. (1995) PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, for the sequence"A-G-T" bonds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between the nucleic acids strands.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g. the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length human EBPH and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but are not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding EBPH orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for northern blotanalysis), cDNA (in solution or bound to a solid support), extract fromcells or a tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is complementary to SEQ ID NO:2 by northern analysis hybridizationassays is indicative of the presence of mRNA encoding EBPH in a sampleand thereby correlates with expression of the transcript from the geneencoding the protein.

"Alterations" in the polynucleotide of SEQ ID NO:2, as used herein,comprise any alteration in the sequence of polynucleotijes encoding EBPHincluding deletions, insertions, and point mutations that may bedetected using hybridization assays. Included within this definition isthe detection of alterations to the genomic DNA sequence which encodesEBPH (e.g., by alterations in the pattern of restriction fragment lengthpolymorphisms capable of hybridizing to SEQ ID NO:2), the inability of aselected fragment of SEQ ID NO:2 to hybridize to a sample of genomic DNA(e.g., using allele-specific oligonucleotide probes), and improper orunexpected hybridization, such as hybridization to a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingEBPH (e.g., using fluorescent in situ hybridization (FISH) to metaphasechromosomes spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind EBPH polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide orpeptide used to immunize an animal can be derived from translated cDNAor synthesized chemically, and can be conjugated to a carrier protein,if desired. Commonly used carriers that are chemically coupled topeptides include bovine serum albumin and thyroglobulin. The coupledpeptide is then used to immunize the animal (e.g., a mouse, a rat, or arabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

THE INVENTION

The invention is based on the discovery of eosinophil-derived basicprotein (EBPH), the polynucleotides encoding EBPH, and the use of thesecompositions for the diagnosis, prevention, or treatment of disordersassociated with excessive eosinophil accumulation and degranulation suchas type I allergic reactions, or disorders in pregnancy related toplacental-derived EBPH.

Nucleic acids encoding the human EBPH of the present invention werefirst identified in cDNA, Incyte Clone 1525913, from an IL-5 stimulatedumbilical cord blood cDNA library (UCMCL5T01), through acomputer-generated search for amino acid sequence alignments. Aconsensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones1525915, 1488589, 1486890, and 1491748, all from the UCMCL5T01 cDNAlibrary.

In one embodiment, the invention encompasses EBPH, a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A1B, and 1C. EBPH is 225 amino acids in length. EBPH has chemical andstructural homology (FIGS. 2A and 2B) with human MBP (GI 34476; SEQ IDNO:3), guinea pig GMBP-1 (GI 220291; SEQ ID NO:4), and GMBP-2 (GI544241; SEQ ID NO:5). In particular, EBPH and MBP share 48% identity,EBPH and GMBP-1 share 47% identity, and EBPH and GMBP-2 share 47%identity. As illustrated by FIGS. 2A, 2B and 3, EBPH is expressed as apreproprotein, with a signal peptide predicted to extend from residues 1to 16, an acidic pro domain predicted to extend from residues 17 to 108,and the basic mature coding region predicted to extend from residues 109to 225. The entire 225 amino acid prepro-EBPH protein coding regioncontains 13 cysteines and no potential N-linked glycosylation sites. Thededuced 116 amino acid mature coding region of EBPH contains 10cysteines. A C-type lectin domain consensus sequence pattern extendsfrom amino acids 200 to 216. The C-type lectin domain structure containstwo disulfide bonds. The predicted isoelectric point (pI) forprepro-EBPH is 4.6 and for mature EBPH is 9.6, assuming the formation oftwo disulfide bonds in the lectin domain.

The sequence identity of MBP, GMBP-1 and GMBP-2 to EBPH decreases in thepro domain coding regions and increases in the mature coding regions.The identity of MBP, GMBP-1 and GMBP-2 to EBPH in the pro-regions is27%, 33%, and 38%, respectively, while the identity in the mature codingregions is 63%, 56%, and 53%, respectively. The decreased identity inthe pro domains may be indicative of different functions for proEBPH andproMBP.

The invention also encompasses EBPH variants. A preferred EBPH variantis one having at least 80%, and more preferably 90%, amino acid sequencesimilarity to the EBPH amino acid sequence (SEQ ID NO:1). A mostpreferred EBPH variant is one having at least 95% amino acid sequencesimilarity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode EBPH.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of EBPH can be used to generate recombinant molecules whichexpress EBPH. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid of SEQ ID NO:2, as shown inFIGS. 1A 1B, and 1C.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding EBPH, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring EBPH, and all such variations are to be consideredas being specifically disclosed.

Although nucleotide sequences which encode EBPH and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring EBPH under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding EBPH or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic expression host in accordance with the frequency with whichparticular codons are utilized by the host. Other reasons forsubstantially altering the nucleotide sequence encoding EBPH and itsderivatives, without altering the encoded amino acid sequences, includethe production of RNA transcripts having more desirable properties, suchas a greater half-life, than transcripts produced from the naturallyoccurring sequence.

The invention also encompasses production of a DNA sequence, or portionsthereof, which encode EBPH and its derivatives, entirely by syntheticchemistry. After production, the synthetic gene may be inserted into anyof the many available DNA vectors and cell systems using reagents thatare well known in the art at the time of the filing of this application.Moreover, synthetic chemistry may be used to introduce mutations into asequence encoding EBPH or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Berger and Kimmel (1987; Methods in Enzymol., Vol. 152, AcademicPress, San Diego, Calif.), and may be used at a defined stringency.

Altered nucleic acid sequences encoding EBPH which are encompassed bythe invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent EBPH. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentEBPH. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of EBPH is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are allelesencoding EBPH. As used herein, an "allele" or "allelic sequence" is analternative form of the gene which may result from at least one mutationin the nucleic acid sequence. Alleles may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one, or many allelic forms. Common mutationalchanges which give rise to alleles are generally ascribed to naturaldeletions, additions, or substitutions of amino acids. Each of thesetypes of changes may occur alone, or in combination with the others, oneor more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The polynucleotide sequence encoding EBPH may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed."restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Gobinda, et al. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO®4.06 primer analysis software (National Biosciences Inc., Plymouth,Minn.). or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and may be useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will contain moresequences which contain the 5' and upstream regions of genes. Use of arandomly primed library may be especially preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into the 5' and 3'non-translated regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotyper™ and Sequence Navigator™ fromPerkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode EBPH, or fusion proteins or functionalequivalents thereof may be used in recombinant DNA molecules to directexpression of EBPH in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressEBPH.

As will be understood by those of skill in the art, it may beadvantageous to produce EBPH-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of EBPH expression or to produce a recombinant RNA transcripthaving desirable properties, such as a half-life which is longer thanthat of a transcript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter the EBPHcoding sequence for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequence. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, to change codon preference, to produce splicevariants, or other mutations, and so forth.

In another embodiment of the invention, a natural, modified, orrecombinant polynucleotide encoding EBPH may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of EBPH activity, it may be useful toencode a chimeric EBPH protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between an EBPH encoding sequence and theheterologous protein sequence, so that the EBPH may be cleaved andpurified away from the heterologous moiety.

In another embodiment, the coding sequence of EBPH may be synthesized,in whole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucd. Acids Res. Symp. Ser. 215-223;Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the EBPH amino acid sequence, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431 A PeptideSynthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of EBPH, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active EBPH, the nucleotide sequenceencoding EBPH or functional equivalents, may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing an EBPH coding sequence andappropriate transcriptional or translational controls. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques, andin vivo recombination or genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express a EBPH coding sequence. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterialexpression vectors (e.g. Ti or pBR322 plasmid): or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene,LaJolla, Calif.) or pSportl (Gibco BRL) and ptrp-lac hybrids, and thelike may be used. The baculovirus polyhedrin promoter may be used ininsect cells. Promoters or enhancers derived from the genomes of plantcells (e.g., heat shock, RUBISCO, and storage protein genes) or fromplant viruses (e.g., viral promoters or leader sequences) may be clonedinto the vector. In mammalian cell systems, promoters from mammaliangenes or from mammalian viruses are preferable. If it is necessary togenerate a cell line that contains multiple copies of the sequenceencoding EBPH, vectors based on SV40 or EBV may be used with anappropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for EBPH. For example, when largequantities of EBPH are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the EBPH coding sequence may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Lleeke, G. et al. (1989) J. Biol.Chem. 264:5503-5509); and the like. pGEX vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast Saccharomvces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel, et al. (supra)and Grant, et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression of asequence encoding EBPH may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu et al. (1987) EMBO J. 6:307-311; Brisson et al. (1984)Nature 310:511-514). Alternatively, plant promoters such as the smallsubunit of RUBISCO or heat shock promoters may be used (Coruzzi et al.(1984) EMBO J. 3:1671-1680; Broglie et al. (1984) Science 224:838-843;Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196 or Weissbachand Weissbach (1988) Methods for Plant Molecular Biology, AcademicPress, New York, N.Y.; pp. 421-463).

An insect system may also be used to express EBPH. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The EBPH coding sequence may be clonedinto a non-essential region of the virus, such as the polyhedrin gene,and placed under control of the polyhedrin promoter. Successfulinsertion of EBPH will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which EBPH may be expressed (Smith et al. (1983) J. Virol46:584; Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci.91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, an EBPH coding sequence may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing EBPH in infected host cells (Logan and Shenk(1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of a EBPH sequence. Such signals include the ATG initiationcodon and adjacent sequences. In cases where sequences encoding EBPH,its initiation codon, and upstream sequences are inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only coding sequence, ora portion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162; Bittner et al. (1987)Methods Enzymol. 153:516-544).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the introduced foreignprotein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressEBPH may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or separate vector. Following theintroduction of the vector, cells may be allowed to grow for 1-2 days inan enriched media before they are switched to selective media. Thepurpose of the selectable marker is to confer resistance to selection,and its presence allows growth and recovery of cells which successfullyexpress the introduced sequences. Resistant clones of stably transformedcells may be proliferated using tissue culture techniques appropriate tothe cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate, GUS, andluciferase and its substrate, luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding EBPH isinserted within a marker gene sequence, recombinant cells containingsequences encoding EBPH can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with anEBPH sequence under the control of a single promoter. Expression of themarker gene in response to induction or selection usually indicatesexpression of the tandem EBPH as well.

Alternatively, host cells which contain the coding sequence for EBPH andexpress EBPH may be identified by a variety of procedures known to thoseof skill in the art. These procedures include, but are not limited to,DNA-DNA or DNA-RNA hybridizations, fluorescent activated cell sortingand protein bioassay or immunoassay techniques which include membrane,solution, or chip based technologies for the detection and/orquantification of the nucleic acid or protein.

The presence of the polynucleotide sequence encoding EBPH can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding EBPH.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the EBPH-encoding sequence todetect transfectants containing DNA or RNA encoding EBPH. As used herein"oligonucleotides" or "oligomers" refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, which can be used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofEBPH, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassa:, (RIA), and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson EBPH is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton etal. (1990, Serological Methods, a Laboratory Manual, APS Press, St Paul,Minn.) and Maddox DE et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding EBPH includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequence encoding EBPH, or anyportion of it, may be cloned into a vector for the production of an mRNAprobe. Such vectors are known in the art, are commercially available,and may be used to synthesize RNA probes in vitro by addition of anappropriate RNA polymerase such as T7, T3. or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia Upjohn, (Kalamazoo, Mich.);Promega (Madison, Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio)).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a nucleotide sequence encoding EBPH may becultured under conditions suitable for the expression and recovery ofthe encoded protein from cell culture. The protein produced by arecombinant cell may be secreted or contained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode EBPH may be designed to contain signal sequences which directsecretion of EBPH through a prokaryotic or eukaryotic cell membrane.Other recombinant constructions may be used to join sequences encodingEBPH to nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and EBPH may be used to facilitate purification. Onesuch expression vector which may be used provides for expression of afusion protein containing EBPH and a nucleic acid encoding 6 histidineresidues followed by thioredoxin and an enterokinase cleavage site. Thehistidine residues facilitate purification on IMIAC (immobilized metalion affinity chromatography as described in Porath, J. et al. (1992;Protein Exp.Purif. 3:263-281) while the enterokinase cleavage siteprovides a means for purifying EBPH from the fusion protein. Adiscussion of vectors which contain fusion proteins is provided inKroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

In addition to recombinant production. fragments of EBPH may be producedby direct peptide synthesis using solid-phase techniques (of Stewart etal. (1969) Solid-Phase Peptide Synthesis, W. H. Freeman Co., SanFrancisco, Calif.; Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154).In vitro protein synthesis may be performed using manual techniques orby automation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Variousfragments of EBPH may be chemically synthesized separately and combinedusing chemical methods to produce the full length molecule.

THERAPEUTICS

In another embodiment of the invention, EBPH or fragments thereof may beused for therapeutic purposes.

Chemical and structural homology exists among EBPH and the eosinophilgranule MBPs from human and guinea pig. In addition, northern analysisdemonstrates that mRNA encoding full-length EBPH was found only in alibrary constructed from pooled human umbilical cord blood cellscultured in the presence of IL-5 (UCMCL5T01). Since umbilical cord bloodis a rich source of stem cells and IL-5 is a potent eosinophilicdifferentiation factor, EBPH is believed to function as an eosinophilgranule protein. Furthermore, a significant proportion of the cDNAclones in the UCMCL5T01 library encode eosinophil-specific proteins. Itmust be noted, however, that naturally occurring expression of EBPH isnot necessarily limited to eosinophils.

From the homology and expression information provided above, it appearsthat EBPH plays a role in host defense mechanisms against parasitic andbacterial infections. Accordingly, in another embodiment of theinvention, EBPH or derivatives thereof may be used as cytolytic agentsin the treatment of such infections.

In another embodiment of the invention, EBPH may be used as a cytolyticagent against cancer cells. Control of EBPH activity as a novel approachto cancer treatment may be especially useful in combination therapy withother, conventional chemotherapeutic agents. Such combinations oftherapeutic agents having different cellular mechanisms of action oftenhave synergistic effects allowing the use of lower effective doses ofeach agent and lessening side effects.

Eosinophil-associated diseases and conditions, including type I allergicreactions, vary in severity. Allergic rhinitis, which affects a largesegment of the population, has severe economic impact. Anaphylaxis is amajor complication of allergic reactions which in many instances isfatal. Asthma is a chronic disease of the airways characterized by mucushypersecretion as well as bronchial inflammation, edema, andhyperresponsiveness resulting in increased bronchoconstriction. Atopicdermatitis is an allergic skin disease associated with high eosinophillevels which result in skin lesions. Hypereosinophilic syndrome andeosinophilic endomyocardial disease may lead to severe cardiac tissuedamage and cardiac failure. Graft-vs.-host disease is also associatedwith high eosinophil accumulation and degranulation.

Therefore, in another embodiment of the invention, vectors expressingantisense and antagonists or inhibitors which block or modulate theeffect of EBPH may be used in those situations where such inhibition ormodulation is therapeutically desirable. Such situations may includediseases and conditions with which eosinophil accumulation and granulerelease are involved, including the diseases discussed above. In suchsituations, EBPH released from eosinophils may promote or exacerbateinflammation and tissue damage. EBPH may also induce further eosinophildegranulation and thus act as an autocrine mediator in the pathogenesisof eosinophil-associated diseases.

Antagonists of EBPH may be produced using methods which are generallyknown in the art. In one aspect, proEBPH may be useful as an inhibitorof EBPH-associated functions, including, but not limited to, thoseassociated with the activation and degranulation of eosinophils,basophils, and neutrophils, including histamine and superoxide release.To prevent the processing of administered proEBPH into mature EBPH invivo variants of EBPH may be produced which contain at least onemutation in SEQ ID NO:1 located at or about the pro-mature domainboundary, said boundary located approximately at amino acid residues 108and 109 of SEQ ID NO:1.

In another aspect of the invention, the isolated EBPH pro domain,comprising amino acid residues 17 to 108 of SEQ ID NO:1 may likewise beuseful as an inhibitor of mature EBPH. The pro domain may beparticularly useful in preventing or treating EBPH-associated tissuedamage. Most particularly, the EBPH pro domain may be administeredtopically to prevent or to treat tissue damage associated with EBPH.Such applications may include, but are not limited to: inhalants for theprevention or treatment of bronchial hyperreactivity and tissue damageassociated with asthma; nose drops for the prevention or treatment ofnasal tissue irritation associated with allergic rhinitis; and ointmentsfor the prevention or treatment of skin lesions associated with atopicdermatitis.

In another aspect, antibodies which are specific for EBPH may be used asan agonist, antagonist, or as part of a targeting or delivery mechanismso as to bring a pharmaceutical agent to cells or tissues which expressEBPH.

The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, and humans, may be immunized by injection with EBPHor any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to EBPH have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of FBPH amino acids may be fused with those of another proteinsuch as keyhole limpet hemocvanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to EBPH may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Koehler et al. (1975) Nature 256:495-497; Kosbor et al.(1983) Immunol. Today 4:72; Cote et al. (1983) Proc. Natl. Acad. Sci.80:2026-2030; Cole et al. (1985) Monoclonal Antibodies and CancerTherapy, Alan R. Liss Inc., New York, N.Y., pp. 77-96).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al. (1984) Proc. Natl.Acad. Sci. 81:6851-6855; Neuberger et al. (1984) Nature 312:604-608;Takeda et al. (1985) Nature 314:452-454). Alternatively, techniquesdescribed for the production of single chain antibodies may be adapted,using methods known in the art, to produce EBPH-specific single chainantibodies. Antibodies with related specificity but of distinctidiotypic composition may be generated by chain shuffling from randomcombinatorial immunoglobulin libraries (Burton D. R. (1991) Proc. Natl.Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for EBPH mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 256:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between EBPH and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a specific EBPH protein is preferred,but a competitive binding assay may also be employed (Maddox (1983),supra).

In another embodiment of the invention, the polynucleotides encodingEBPH, or any fragment thereof or antisense sequences, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding EBPH may be used in situations in which it would be desirableto block the synthesis of the protein. In particular, cells may betransformed with antisense sequences to polynucleotides encoding EBPH.Thus, antisense sequences may be used to prevent EBPH-associated tissuedamage, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers, or largerfragments, can be designed from various locations along the coding orcontrol regions.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensepolynucleotides of the gene encoding EBPH. See, for example, thetechniques described in Sambrook et al. (supra) and Ausubel et al.(supra).

Genes encoding EBPH can be turned off by transfecting a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes EBPH. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until all copies are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding EBPH, i.e. the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions -10 and +10 from the ATG start site, are preferred.The antisense molecules may also be designed to block translation ofmRNA by preventing the transcript from binding to ribosomes. Similarly,inhibition can be achieved using "triple helix" base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.).

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding EBPH.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of RNA molecules. Theseinclude techniques for chemically synthesizing oligonucleotides such assolid phase phosphoramidite chemical synthesis. Alternatively, RNAmolecules may be generated by in vitro and in vivo transcription of DNAsequences encoding EBPH. Such DNA sequences may be incorporated into awide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, antisense cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Methods for introducing vectors into cells or tissues include thosemethods discussed above. These methods are equally suitable for use invivo, in vitro, and ex vivo. For ex vivo therapy, vectors may beintroduced into stem cells taken from the patient and clonallypropagated for autologous transplant back into that same patient.Delivery by transfection and by liposome injections may be achievedusing methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysuitable subject including, for example, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of EBPH, antibodies toEBPH, mimetics, agonists, antagonists, or inhibitors of EBPH. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers, well known in the art, indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycol with or withoutstabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1 mM-50 mM histidine, 0.1%-2% sucrose, and 2%-7% mannitolat a pH range of 4.5 to 5.5 that is/are combined with buffer prior touse.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of EBPH, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example EBPH or fragments thereof, agonists antibodiesto EBPH or agonists, antagonists, or inhibitors of EBPH, whichameliorates the symptoms or condition. Therapeutic efficacy and toxicitymay be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., ED50 (the dose therapeutically effectivein 50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject who requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combinations, reaction sensitivities,and tolerance/response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, or onceevery two weeks depending on half-life and clearance rate of theparticular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which are specific for EBPH may beused for the diagnosis of conditions or diseases characterized byexpression of EBPH, or in assays to monitor patients being treated withEBPH, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prenared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for EBPH includemethods which utilize the antibody and a label to detect EBPH in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring EBPHare known in the art and provide a basis for diagnosing altered orabnormal levels of EBPH expression. Normal or standard values for EBPHexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toEBPH under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods,preferably by photometric means. Quantities of EBPH expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingEBPH may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofEBPH may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of EBPH,and to monitor regulation of EBPH levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding EBPH or closely related molecules, may be used to identifynucleic acid sequences which encode EBPH. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding EBPH, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthese EBPH encoding sequences. The hybridization probes of the subjectinvention may be derived from the nucleotide sequence of SEQ ID NO:2 orfrom genomic sequence including promoter, enhancer elements, and intronsof the naturally occurring EBPH.

Other means for producing specific hybridization probes for DNAsencoding EBPH include the cloning of nucleic acid sequences encodingEBPH or EBPH derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate radioactively labelednucleotides. Hybridization probes may be labeled by a variety ofreporter groups, for example, radionuclides such as 32P or 35S, orenzymatic labels, such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems, and the like.

Polynucleotide sequences encoding EBPH may be used for the diagnosis ofconditions or diseases which are associated with expression of EBPH.Examples of such conditions or diseases include type I allergicreactions, parasitic infections, and eosinophilias. In addition, proEBPHin placenta may be complexed with other pregnancy-associated moleculessuch as PAPP-A and angiotensinogen and thus may be useful in thediagnosis of pregnancy-associated conditions. The polynucleotidesequences encoding EBPH may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indip stick, pin, chip, and ELISA assays of fluids or tissues from patientbiopsies to detect altered EBPH expression. Such qualitative orquantitative methods are well known in the art.

In order to provide a basis for the diagnosis of disease associated withexpression of EBPH, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes EBPH, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects witha dilution series of EBPH measured in the same experiment, where a knownamount of a substantially purified EBPH is used. Standard valuesobtained from normal samples may be compared with values obtained fromsamples from patients who are symptomatic for disease associated withEBPH. Deviation between standard and subject values is used to establishthe presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

Additional diagnostic uses for oligonucleotides encoding EBPH mayinvolve the use of PCR. Such oligomers may be chemically synthesized,generated enzymatically, or produced from a recombinant source.Oligomers will preferably consist of two nucleotide sequences, one withsense orientation (5'→3') and another with antisense (3'←5'), employedunder optimized conditions for identification of a specific gene orcondition. The same two oligomers, nested sets of oligomers, or even adegenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of EBPHinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236.) The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor calorimetric response gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequence whichencodes EBPH may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequence may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual ofBasic Techniques, Pergamon Press, New York, N.Y.) may be correlated withother physical chromosome mapping techniques and genetic map data.Examples of genetic map data can be found in the 1994 Genome Issue ofScience (265:1981f). Correlation between the location of the geneencoding EBPH on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti et al.(1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, EBPH, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such a test may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenEBPH and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to EBPH, large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with EBPH, or fragments thereof, and washed. Bound EBPH is thendetected by methods well known in the art. Purified EBPH can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding EBPH specificallycompete with a test compound for binding EBPH. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with EBPH.

In additional embodiments, the nucleotide sequences which encode EBPHmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I Construction of cDNA Libraries

Mononuclear cells were obtained from the umbilical cord blood of 12individuals and were cultured in the presence of IL-5 for 12 days. Thecells were homogenized and lysed using a Brinkmann Homogenizer PolytronPT-3000 (Brinkmann Instruments, Westbury, N.J.) in guanidiniumisothiocyanate solution. The lysate was centrifuged over a 5.7 M CsCIcushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge(Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.The RNA was extracted with acid phenol pH 4.7, precipitated using 0.3 Msodium acetate and 2.5 volumes of ethanol, resuspended in RNAse freewater, and DNAse treated at 37° C. The RNA extraction was repeated withacid phenol pH 4.7 and precipitated with sodium acetate and ethanol asbefore. The mRNA was then isolated using the Qiagen Oligotex kit(QIAGEN, Inc., Chatsworth, Calif.) and used to construct the cDNAlibrary.

The mRNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat.#18248-013, Gibco/BRL).

The commercial plasmid pSPORT 1™ (Gibco/BRL) was digested with EcoRIrestriction enzyme (New England Biolabs, Beverley, Mass.). Theoverhanging ends of the plasmid were filled in using Klenow enzyme (NewEngland Biolabs) and 2'-deoxynucleotide 5'-triphosphates (dNTPs). Theplasmid was self-ligated and transformed into the bacterial host, E.coli strain JM 109. An intermediate plasmid produced by the bacteriafailed to digest with EcoR I confirming the desired loss of the EcoR Irestriction site.

This intermediate plasmid (pSPORT 1-ΔRI) was then digested with Hind IIIrestriction enzyme (New England Biolabs) and the overhang was filled inwith Klenow and dNTPs. A 10-mer linker of sequence 5'. . . CGGAATTCCG .. . 3' was phosphorylated and ligated onto the blunt ends. The productof the ligation reaction was digested with EcoR I and self-ligated.Following transformation into JM109 host cells plasmids were isolatedand screened for the digestibility with EcoR I but not with Hind III. Asingle colony which met this criteria was designated pINCY 1. Theplasmid produced by this colony was sequenced and found to containseveral copies of the 10-mer linker. These extra linkers did not presenta problem as they were eliminated when the vector was prepared forcloning.

The plasmid was tested for its ability to incorporate cDNAs from alibrary prepared using Not I and EcoR I restriction enzymes. Severalclones were sequenced and a single clone containing an insert ofapproximately 0.8 kb was selected to prepare a large quantity of theplasmid for library production. After digestion with Not I and EcoR I,the plasmid and the cDNA insert were isolated on an agarose gel and thevector was purified on a QIAQuick-™ (QIAGEN, Inc.) column for use inlibrary construction.

cDNAs were fractionated on a Sepharose CL4B column (Cat. #275105-01,Pharmacia), and those cDNAs exceeding 400 bp were ligated into pSport I.The plasmid pSport I was subsequently transformed into DH5a™ competentcells (Cat. #18258-012, Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 Plasmid Kit for Rapid Extraction Alkaline Lysis Plasmid Minipreps(Catalog #26173, QIAGEN, Inc.). This kit enabled the simultaneouspurification of 96 samples in a 96-well block using multi-channelreagent dispensers. The recommended protocol was employed except for thefollowing changes: 1) the bacteria were cultured in 1 ml of sterileTerrific Broth (Catalog #22711, Life Technologies) with carbenicillin at25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems; andthe reading frame was determined.

Most of the sequences disclosed herein were sequenced according tostandard ABI protocols, using ABI kits (Cat. Nos. 79345, 79339, 79340,79357, 79355). The solution volumes were used at 0.25×-1.0×concentrations. Some of the sequences disclosed herein were sequencedusing different solutions and dyes which, unless otherwise noted, camefrom Amersham Life Science (Cleveland, Ohio).

Stock solutions were prepared with HPLC water. The following solutionswere each mixed by vortexing for 2 min: 1) Tris-EDTA (TE) Buffer wasprepared by adding 49 ml water to 1 ml 50× Tris-EDTA concentrate, and 2)10% Reaction Buffer was prepared by adding 45 ml water to 5 mlConcentrated Thermo Sequenase (TS) Reaction Buffer.

Energy transfer (ET) primers (0.2 μM) were prepared in the followingmanner. Each primer tube was centrifuged prior to opening to assure thatall primer powder was on the bottom of the tube. After eachsolubilization step, the mixture was vortexed for 2 min and thencentrifuged for about 10 sec in a table-top centrifuge. 1 ml of 1×TE wasadded to each primer powder; adenine and cytosine dissolved primers(5-carboxyrhodamine-6G (R6G) and 6-carboxyfluorescein (FAM),respectively), were diluted with 9 ml 1×TE. Guanine and thymine dyes(N,N,N',N"-tetramethyl-6-carboxyrhodamine (TAM) and6-carboxy-X-rhodamine (ROX), respectively) were diluted with 19 ml 1×TE.

The sequencing reaction ready mix was prepared as follows: 1)nucleotides A and C (8 ml of each) were added to 6 ml ET primer and 18ml TS reaction buffer; and 2) nucleotides G and T (8 ml of each) wereadded to 6 ml ET primer and 18 ml TS reaction buffer. After vortexingfor 2 min and centrifuging for 20 sec, the resulting solution wasdivided into tubes in volumes of 8 ml per tube in order to make 1×(A,C)and 2×(G,T) solutions.

Prior to thermal cycling, each nucleotide was individually mixed withDNA template in the following proportions:

    ______________________________________                                        Reagent      A (μL)                                                                             C (μL) G (μL)                                                                           T (μL)                                ______________________________________                                        Reaction ready premix                                                                      2       2         4     4                                          DNA template 1 1 2 2                                                          Total volume 3 3 6 6                                                        ______________________________________                                    

These solutions were subjected to the usual thermal cycling:

1. Rapid thermal ramp to 94° C. (94° C. for 20 sec)*

2. Rapid thermal ramp to 50° C. (50° C. for 40 sec)*

3. Rapid thermal ramp to 68° C. (68° C. for 60 sec)*

* Steps 1, 2, and 3 were repeated for 15 cycles

4. Rapid thermal ramp to 94° C. (94° C. for 20 sec)**

5. Rapid thermal ramp to 68° C. (68° C. for 60 sec)**

** Steps 4 and 5 were repeated for 15 cycles

6. Rapid thermal ramp to 4° C. and hold until ready to combine.

After thermal cycling, the A, C, G, and T reactions were combined witheach DNA template. Then, 50 μL 100% ethanol was added and the solutionwas spun at 4° C. for 30 min. The supernatant was decanted and thepellet was rinsed with 100 μL 70% ethanol. After being spun for 15 minthe supernatant was discarded and the pellet was dried for 15 min undervacuum. The DNA sample was dissolved in 3 μL of formnaldehyde/50 mMEDTA. The resulting samples were loaded on wells in volumes of 2 μL perwell for sequencing in ABI sequencers.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT™ 670Sequence Analysis System. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles, Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT-670 Sequence Analysis System using the methods similar to thoseused in DNA sequence homologies. Pattern Specification Language andparameter windows were used to search protein databases for sequencescontaining regions of homology which were scored with an initial value.Dot-matrix homology plots were examined to distinguish regions ofsignificant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul SF(1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.215:403-410), was used to search for local sequence alignments. BLASTproduces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul S. F. 1993 and 1990,supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals, Inc.). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding EBPH occurs. Abundance and percentageabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercentage abundance is abundance divided by the total number ofsequences examined in the cDNA library.

V Extension of EBPH-Encoding Polynucleotides to Full Length or toRecover Regulatory Elements

Full length EBPH-encoding nucleic acid sequence (SEQ ID NO:2) is used todesign oligonucleotide primers for extending a partial nucleotidesequence to full length or for obtaining 5' sequences from genomiclibraries. One primer is synthesized to initiate extension in theantisense direction (XLR) and the other is synthesized to extendsequence in the sense direction (XLF). Primers are used to facilitatethe extension of the known sequence "outward" generating ampliconscontaining new, unknown nucleotide sequence for the region of interest.The initial primers are designed from the cDNA using OLIGO® 4.06(National Biosciences), or another appropriate program, to be 22-30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desires, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit. PCR isperformed using the Peltier Thermal Cycler (PTC200; M. J. Research,Watertown, Mass.) and the following parameters:

Step 1 94° C. for 1 min (initial denaturation)

Step 2 65° C. for 1 min

Step 3 68° C. for 6 min

Step 4 94° C. for 15 sec

Step 5 65° C. for 1 min

Step 6 68° C. for 7 min

Step 7 Repeat step 4-6 for 15 additional cycles

Step 8 94° C. for 15 sec

Step 9 65° C. for 1 min

Step 10 68° C. for 7:15 min

Step 11 Repeat step 8-10 for 12 cycles

Step 12 72° C. for 8 min

Step 13 4° C. (and holding)

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products are selected and removed from the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQuick™ (QIAGEN Inc. Chatsworth, Calif.). After recovery ofthe DNA, Klenow enzyme is used to trim single-stranded, nucleotideoverhangs creating blunt ends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

Step 1 94° C. for 60 sec

Step 2 94° C. for 20 sec

Step 3 55° C. for 30 sec

Step 4 72° C. for 90 sec

Step 5 Repeat steps 2-4 for an additional 29 cycles

Step 6 72° C. for 180 sec

Step 7 4° C. (and holding)

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 mCi of [γ-³² P] adenosine triphosphate(Amersham, Chicago, Ill.) and T4 polynucleotide kinase (DuPont NEN®,Boston, Mass.). The labeled oligonucleotides are substantially purifiedwith Sephadex G-25 superfine resin column (Pharmacia Upjohn). A portioncontaining 10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots or the blots areexposed in a PhoshorImager cassette (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

VII Antisense Molecules

Antisense molecules to the EBPH-encoding sequence, or any part thereof,is used to inhibit in vivo or in vitro expression of naturally occurringEBPH. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequences of EBPH, as shown in FIGS. 1A 1B, and 1C is used to inhibitexpression of naturally occurring EBPH. The complementaryoligonucleotide is designed from the most unique 5' sequence as shown inFIGS. 1A 1B, and 1C and used either to inhibit transcription bypreventing promoter binding to the upstream noritranslated sequence ortranslation of an EBPH-encoding transcript by preventing the ribosomefrom binding. Using an appropriate portion of the signal and 5' sequenceof SEQ ID NO:2, an effective antisense oligonucleotide includes any15-20 nucleotides spanning the region which translates into the signalor 5' coding sequence of the polypeptide as shown in FIGS. 1A 1B, and1C.

VIII Expression of EBPH

Expression of EBPH is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express EBPH in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofEBPH into the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of EBPH Activity

The cytolytic activity of EBPH is assayed by monitoring the release of⁵¹ Cr from cells treated with EBPH. Bronchial epithelial cells, or othersuitable cells, are incubated with ⁵¹ Cr (Amersham) in an appropriatemedium for 1 hour at 37° C. The cells are washed to removeunincorporated ⁵¹ Cr and are resuspended. The cytolysis reaction isinitiated by addition of EBPH followed by incubation for a predeterminedlength of time at 37° C. The reaction mixture is centrifuged at 4° C.,and radioactivity of an aliquot of the cell-free supernatant is assayedin a gamma scintillation counter. Total cellular ⁵¹ Cr content isdetermined with an aliquot of the reaction mixture lysed in 0.04% TritonX-100, and spontaneous ⁵¹ Cr release is determined for cells incubatedunder the same conditions but in the absence of EBPH.

X Production of EBPH Specific Antibodies

EBPH that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431 A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) byreaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS;Ausubel et al., supra). Rabbits are immunized with the oligopeptide-KLHcomplex in complete Freund's adjuvant. The resulting antisera are testedfor antipeptide activity, for example, by binding the peptide toplastic, blocking with 1% BSA, reacting with rabbit antisera, washing,and reacting with radioiodinated, goat anti-rabbit IgG.

XI Purification of Naturally Occurring EBPH Using Specific Antibodies

Naturally occurring or recombinant EBPH is substantially purified byimmunoaffinity chromatography using antibodies specific for EBPH. Animmunoaffinity column is constructed by covalently coupling EBPHantibody to an activated chromatographic resin, such as CnBr-activatedSepharose (Pharmacia Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing EBPH is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof EBPH (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/EBPH binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and EBPH is collected.

XII Identification of Molecules Which Interact with EBPH

EBPH or biologically active fragments thereof are labeled with 125Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled EBPH, washed and any wells withlabeled EBPH complex are assayed. Data obtained using differentconcentrations of EBPH are used to calculate values for the number,affinity, and association of EBPH with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 5                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 225 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY:                                                                  (B) CLONE: Consensus                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Gln Arg Leu Leu Leu Leu Pro Phe Leu Le - #u Leu Gly Thr Val        Ser                                                                              1               5  - #                10  - #                15              - - Ala Leu His Leu Glu Asn Asp Ala Pro His Le - #u Glu Ser Leu Glu Thr                  20      - #            25      - #            30                   - - Gln Ala Asp Leu Gly Gln Asp Leu Asp Ser Se - #r Lys Glu Gln Glu Arg              35          - #        40          - #        45                       - - Asp Leu Ala Leu Thr Glu Glu Val Ile Gln Al - #a Glu Gly Glu Glu Val          50              - #    55              - #    60                           - - Lys Ala Ser Ala Cys Gln Asp Asn Phe Glu As - #p Glu Glu Ala Met Glu      65                  - #70                  - #75                  - #80        - - Ser Asp Pro Ala Ala Leu Asp Lys Asp Phe Gl - #n Cys Pro Arg Glu Glu                      85  - #                90  - #                95               - - Asp Ile Val Glu Val Gln Gly Ser Pro Arg Cy - #s Lys Thr Cys Arg Tyr                  100      - #           105      - #           110                  - - Leu Leu Val Arg Thr Pro Lys Thr Phe Ala Gl - #u Ala Gln Asn Val Cys              115          - #       120          - #       125                      - - Ser Arg Cys Tyr Gly Gly Asn Leu Val Ser Il - #e His Asp Phe Asn Phe          130              - #   135              - #   140                          - - Asn Tyr Arg Ile Gln Cys Cys Thr Ser Thr Va - #l Asn Gln Ala Gln Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Trp Ile Gly Gly Asn Leu Arg Gly Trp Phe Le - #u Trp Lys Arg Phe        Cys                                                                                             165  - #               170  - #               175             - - Trp Thr Asp Gly Ser His Trp Asn Phe Ala Ty - #r Trp Ser Pro Gly Gln                  180      - #           185      - #           190                  - - Pro Gly Asn Gly Gln Gly Ser Cys Val Ala Le - #u Cys Thr Lys Gly Gly              195          - #       200          - #       205                      - - Tyr Trp Arg Arg Ala Gln Cys Asp Lys Gln Le - #u Pro Phe Val Cys Ser          210              - #   215              - #   220                          - - Phe                                                                      225                                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 865 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY:                                                                  (B) CLONE: Consensus                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GACGGCTCGA GTGGAGGTCT CAGACTCTTG GAAGGGGCTA TACTAGACAC AC -             #AAAGACAG     60                                                                 - - CCCCAAGAAG GACGGTGGAG TAGTGTCCTC GCTAAAAGAC AGTAGATATG CA -            #ACGCCTCT    120                                                                 - - TGCTCCTGCC CTTTCTCCTG CTGGGAACAG TTTCTGCTCT TCATCTGGAG AA -            #TGATGCCC    180                                                                 - - CCCATCTGGA GAGCCTAGAG ACACAGGCAG ACCTAGGCCA GGATCTGGAT AG -            #TTCAAAGG    240                                                                 - - AGCAGGAGAG AGACTTGGCT CTGACGGAGG AGGTGATTCA GGCAGAGGGA GA -            #GGAGGTCA    300                                                                 - - AGGCTTCTGC CTGTCAAGAC AACTTTGAGG ATGAGGAAGC CATGGAGTCG GA -            #CCCAGCTG    360                                                                 - - CCTTAGACAA GGACTTCCAG TGCCCCAGGG AAGAAGACAT TGTTGAAGTG CA -            #GGGAAGTC    420                                                                 - - CAAGGTGCAA GACCTGCCGC TACCTATTGG TGCGGACTCC TAAAACTTTT GC -            #AGAAGCTC    480                                                                 - - AGAATGTCTG CAGCAGATGC TACGGAGGCA ACCTTGTCTC TATCCATGAC TT -            #CAACTTCA    540                                                                 - - ACTATCGCAT TCAGTGCTGC ACTAGCACAG TCAACCAAGC CCAGGTCTGG AT -            #TGGAGGCA    600                                                                 - - ACCTCAGGGG CTGGTTCCTG TGGAAGCGGT TTTGCTGGAC TGATGGGAGC CA -            #CTGGAATT    660                                                                 - - TTGCTTACTG GTCCCCAGGG CAACCTGGGA ATGGGCAAGG CTCCTGTGTG GC -            #CCTATGCA    720                                                                 - - CCAAAGGAGG TTATTGGCGA CGAGCTCAAT GCGACAAGCA ACTGCCCTTC GT -            #CTGCTCCT    780                                                                 - - TCTAAGCCAG CGGCACGGAG ACCCTGCCAG CAGCTCCCTC CCGTCCCCCA AC -            #CTCTCCTG    840                                                                 - - CTCATAAATC CAGACTTCCC ACAGC          - #                  - #                  865                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 222 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 34476                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Lys Leu Pro Leu Leu Leu Ala Leu Leu Ph - #e Gly Ala Val Ser Ala       1               5  - #                10  - #                15               - - Leu His Leu Arg Ser Glu Thr Ser Thr Phe Gl - #u Thr Pro Leu Gly Ala                  20      - #            25      - #            30                   - - Lys Thr Leu Pro Glu Asp Glu Glu Thr Pro Gl - #u Gln Glu Met Glu Glu              35          - #        40          - #        45                       - - Thr Pro Cys Arg Glu Leu Glu Glu Glu Glu Gl - #u Trp Gly Ser Gly Ser          50              - #    55              - #    60                           - - Glu Asp Ala Ser Lys Lys Asp Gly Ala Val Gl - #u Ser Ile Ser Val Pro      65                  - #70                  - #75                  - #80        - - Asp Met Val Asp Lys Asn Leu Thr Cys Pro Gl - #u Glu Glu Asp Thr Val                      85  - #                90  - #                95               - - Lys Val Val Gly Ile Pro Gly Cys Gln Thr Cy - #s Arg Tyr Leu Leu Val                  100      - #           105      - #           110                  - - Arg Ser Leu Gln Thr Phe Ser Gln Ala Trp Ph - #e Thr Cys Arg Arg Cys              115          - #       120          - #       125                      - - Tyr Arg Gly Asn Leu Val Ser Ile His Asn Ph - #e Asn Ile Asn Tyr Arg          130              - #   135              - #   140                          - - Ile Gln Cys Ser Val Ser Ala Leu Asn Gln Gl - #y Gln Val Trp Ile Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Arg Ile Thr Gly Ser Gly Arg Cys Arg Ar - #g Phe Gln Trp Val        Asp                                                                                             165  - #               170  - #               175             - - Gly Ser Arg Trp Asn Phe Ala Tyr Trp Ala Al - #a His Gln Pro Trp Ser                  180      - #           185      - #           190                  - - Arg Gly Gly His Cys Val Ala Leu Cys Thr Ar - #g Gly Gly Tyr Trp Arg              195          - #       200          - #       205                      - - Arg Ala His Cys Leu Arg Arg Leu Pro Phe Il - #e Cys Ser Tyr                  210              - #   215              - #   220                          - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 233 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 220291                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Met Lys Leu Leu Leu Leu Leu Ala Leu Leu Le - #u Gly Ala Val Ser Thr       1               5  - #                10  - #                15               - - Arg His Leu Lys Val Asp Thr Ser Ser Leu Gl - #n Ser Leu Arg Gly Glu                  20      - #            25      - #            30                   - - Glu Ser Leu Ala Gln Asp Gly Glu Thr Ala Gl - #u Gly Ala Thr Arg Glu              35          - #        40          - #        45                       - - Ala Thr Ala Gly Ala Leu Met Pro Leu Pro Gl - #u Glu Glu Glu Met Glu          50              - #    55              - #    60                           - - Gly Ala Ser Gly Ser Glu Asp Asp Pro Glu Gl - #u Glu Glu Glu Glu Glu      65                  - #70                  - #75                  - #80        - - Glu Glu Val Glu Phe Ser Ser Glu Leu Asp Va - #l Ser Pro Glu Asp Ile                      85  - #                90  - #                95               - - Gln Cys Pro Lys Glu Glu Asp Thr Val Lys Ph - #e Phe Ser Arg Pro Gly                  100      - #           105      - #           110                  - - Tyr Lys Thr Arg Gly Tyr Val Met Val Gly Se - #r Ala Arg Thr Phe Asn              115          - #       120          - #       125                      - - Glu Ala Gln Trp Val Cys Gln Arg Cys Tyr Ar - #g Gly Asn Leu Ala Ser          130              - #   135              - #   140                          - - Ile His Ser Phe Ala Phe Asn Tyr Gln Val Gl - #n Cys Thr Ser Ala Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Asn Val Ala Gln Val Trp Ile Gly Gly Gl - #n Leu Arg Gly Lys        Gly                                                                                             165  - #               170  - #               175             - - Arg Cys Arg Arg Phe Val Trp Val Asp Arg Th - #r Val Trp Asn Phe Ala                  180      - #           185      - #           190                  - - Tyr Trp Ala Arg Gly Gln Pro Trp Gly Gly Ar - #g Gln Arg Gly Arg Cys              195          - #       200          - #       205                      - - Val Thr Leu Cys Ala Arg Gly Gly His Trp Ar - #g Arg Ser His Cys Gly          210              - #   215              - #   220                          - - Lys Arg Arg Pro Phe Val Cys Thr Tyr                                      225                 2 - #30                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 234 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 544241                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met Lys Leu Leu Leu Leu Leu Ala Leu Leu Va - #l Gly Ala Val Ser Thr       1               5  - #                10  - #                15               - - Arg His Leu Asn Val Asp Thr Ser Ser Leu Gl - #n Ser Leu Gln Gly Glu                  20      - #            25      - #            30                   - - Glu Ser Leu Ala Gln Asp Gly Glu Thr Ala Gl - #u Gly Ala Thr Arg Glu              35          - #        40          - #        45                       - - Ala Ala Ser Gly Val Leu Met Pro Leu Arg Gl - #u Glu Val Lys Glu Glu          50              - #    55              - #    60                           - - Met Glu Gly Gly Ser Gly Ser Glu Asp Asp Pr - #o Glu Glu Glu Glu Glu      65                  - #70                  - #75                  - #80        - - Glu Lys Glu Met Glu Ser Ser Ser Glu Leu As - #p Met Gly Pro Glu Asp                      85  - #                90  - #                95               - - Val Gln Cys Pro Lys Glu Glu Asp Ile Val Ly - #s Phe Glu Gly Ser Pro                  100      - #           105      - #           110                  - - Gly Cys Lys Ile Cys Arg Tyr Val Val Leu Se - #r Val Pro Lys Thr Phe              115          - #       120          - #       125                      - - Lys Gln Ala Gln Ser Val Cys Gln Arg Cys Ph - #e Arg Gly Asn Leu Ala          130              - #   135              - #   140                          - - Ser Ile His Ser Tyr Asn Ile Asn Leu Gln Va - #l Gln Arg Ser Ser Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ile Leu Asn Val Ala Gln Val Trp Ile Gly Gl - #y Gln Leu Arg Gly        Lys                                                                                             165  - #               170  - #               175             - - Gly His His Lys His Phe His Trp Val Asp Gl - #y Thr Leu Trp Asn Phe                  180      - #           185      - #           190                  - - Trp Tyr Trp Ala Ala Gly Gln Pro Trp Arg Gl - #y Asn Asn Ser Gly Arg              195          - #       200          - #       205                      - - Cys Val Thr Leu Cys Ala Arg Gly Gly His Tr - #p Arg Arg Ser His Cys          210              - #   215              - #   220                          - - Gly Val Arg Arg Ala Phe Ser Cys Ser Tyr                                  225                 2 - #30                                                  __________________________________________________________________________

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence of SEQ ID NO:1.
 2. A composition comprising the isolatedpolypeptide of claim 1 in conjunction with a pharmaceutically acceptablecarrier.
 3. An isolated polypeptide comprising the prodomain, amino acidresidues 17-108, and mature protein domain, amino acid residues 109-225,of SEQ ID NO:1.
 4. A composition comprising the isolated polypeptide ofclaim 3 in conjunction with a pharmaceutically acceptable carrier.
 5. Anisolated polypeptide comprising the mature protein domain, amino acidresidues 109-225, of SEQ ID NO:1.
 6. A composition comprising theisolated polypeptide of claim 5 in conjunction with a pharmaceuticallyacceptable carrier.